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Visualization of Intracellular Tyrosinase Activity in vitro
体外试验中胞内络氨酸酶活性的可视化   

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Riddhi Atul JaniRiddhi Atul Jani*Sudeshna NagSudeshna Nag*Subba Rao Gangi SettySubba Rao Gangi Setty  (*contributed equally to this work)
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Abstract

Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels result in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we describe a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.

Keywords: Tyrosinase(酪氨酸酶), Melanosome(黑素小体), Oculocutaneous albinism(眼皮肤白化病), Copper(铜), L-DOPA(左旋多巴)

Materials and Reagents

  1. Glass coverslips (diameter-12 mm, No.1) (Polar Industrial Corporation, Blue Star, catalog number: 12mm Circular )
    Note: See Recipes for acid wash and sterilization.
  2. Micro slides (L-75 mm x W-25 mm x h-1.35 mm) (Polar Industrial Corporation, Blue Star, catalog number: PIC-1 )
  3. Plastic tissue culture (6 well) plate (Corning, catalog number: 3506 ) and bottle-top vacuum filter (pore size 0.22 μm) (Corning, catalog number: 430015 )
  4. Melanocytes (Immortal wild type mouse melanocytes, melan-Ink4a-Arf-1 from C57BL/6J mice, referred to here as melan-Ink4a) [Resource: The Wellcome Trust Functional Genomics Cell Bank (Sviderskaya et al., 2010)]
  5. Copper(II) sulphate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C7631 )
  6. 3, 4-Dihydroxy-D-phenylalanine (D-DOPA) (Sigma-Aldrich, catalog number: D9378 )
  7. 3, 4-Dihydroxy-L-phenylalanine (L-DOPA) (Sigma-Aldrich, catalog number: D9628 )
  8. HCl (Sigma-Aldrich, catalog number: H1758 )
  9. HNO3 (Merck Millipore, catalog number: 101799 )
  10. KCl (Sigma-Aldrich, catalog number: P9541 )
  11. KH2PO4 (Merck Millipore, catalog number: 104873 )
  12. NaCl (Fisher Scientific, catalog number: BP358-1 )
  13. Na2HPO4·2H2O (Fisher Scientific, catalog number: S472-500 )
  14. Ethanol (70%) (Merck Millipore, catalog number: 818760 )
  15. Fetal bovine serum (Biowest, catalog number: S1810-500 )
  16. Formaldehyde (36.5-38% in H2O) solution (HCHO solution) (Sigma-Aldrich, catalog number: F8775 )
  17. Fluromount-G or mounting medium (SouthernBiotech, catalog number: 0100-01 )
  18. Matrigel Matrix (Corning Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free) (Corning, catalog number: 356231 )
  19. Penicillin-Streptomycin (antibiotic) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  20. RPMI-1640 media (Thermo Fisher Scientific, GibcoTM, catalog number: 31800-022 )
  21. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  22. 0.1% DOPA solution (see Recipes)
  23. 4% Formaldehyde solution (see Recipes)
  24. Growth media (see Recipes)
  25. 0.1% Matrigel matrix solution (see Recipes)
  26. 1x PBS (see Recipes)

Equipment

  1. CO2 incubator (maintained at 37 °C, 10% CO2) (Thermo Fisher Scientific, model: Forma Water Jacketed CO2 incubator )
  2. Forceps (sterilized by autoclave)
  3. Bright field microscope (Olympus Corporation, model: IX81 motorized inverted fluorescence microscope )
  4. Hot-air-oven (Eyela, catalog number: NDO-420W )
  5. Glass beaker (250 ml) (Borosil, catalog number: 1000D21 )

Procedure

  1. Prepare coverslips for the following treatments: 1x PBS and/or D-DOPA (negative control), L-DOPA and L-DOPA with copper. For this, add four sterile coverslips to four wells of 6 well plate using forceps.
    Note: Copper is essential for tyrosinase activity in melanocytes and addition of copper to the fixed cells will allow visualizing tyrosinase activity in the secretory compartments such as endosomes and Golgi and enhance its activity in the melanosomes (Setty et al., 2008).
  2. Coat the coverslips with a thin layer of Matrigel using 0.1% Matrigel matrix solution and dry the coverslips at room temperature for 15-30 min.
  3. Wash the coverslips thrice with 1x PBS (2-3 ml each, room temperature).
  4. Seed the mouse melanocytes approximately 0.5-0.8 x 106 in each well of 6 well plate or 60-70% confluence in any dish and incubate at 37 °C in 10% CO2 incubator.
  5. Fix the cells after 24 h with 4% formaldehyde in 1x PBS (room temperature) for 30 min.
  6. Wash the cells thrice with 1x PBS (room temperature).
  7. Incubate the cells with the following freshly prepared solutions at 37 °C for 2 h.
    a. 1x PBS and/or 0.1% D-DOPA in 1x PBS (negative control)
    b. 0.1% L-DOPA in 1x PBS
    c. 0.1% L-DOPA in 1x PBS with 20 μM copper sulphate (co-factor for tyrosinase)
  8. Remove the solutions and repeat the incubation with the respective solutions.
    Note: L-DOPA in PBS or water undergoes auto-oxidation and turns black after few hours. Moreover, this oxidation results in loss of active L-DOPA substrate in the solution and thus, repetition of this step with freshly made solutions will improve the activity of tyrosinase.
  9. Wash the cells thrice with 1x PBS.
  10. Mount the coverslips with mounting medium on glass slides and image under the bright field microscope (Figure 1).
  11. Keep the bright field exposure time and settings identical for all the samples (Figure 1).
  12. Compare the bright field images of L-DOPA or L-DOPA+copper treated melanocytes with 1x PBS or D-DOPA. The images can be analyzed for differences in signal intensity and number of pigment granules, as well as their distribution within the cells.
    Note: The increase in pigment granule number and intensity in melanocytes treated with L-DOPA or L-DOPA+copper (observe higher granule intensity compared to L-DOPA) is indicative to the presence of active tyrosinase in both intracellular vesicles and endocytic organelles including melanosomes (Setty et al., 2008).

Representative data


Figure 1. In vitro intracellular tyrosinase activity in wild type mouse melanocytes. Freshly fixed mouse melanocytes on glass coverslips were incubated with 1x PBS, D-DOPA (negative control) or L-DOPA with or without copper sulphate (20 μM) for 4 h. Images were captured at identical camera setting in a bright field mode on inverted fluorescence microscope. Arrows indicate the increased tyrosinase activity in cells treated with L-DOPA (with or without copper). Arrowheads point to the regions with enhanced tyrosinase activity in cells treated with L-DOPA+copper. Scale bars, 10 μm and insets, 2.5 times of the white boxed areas. Note that the melanin deposits are increased in cells treated with L-DOPA (with or without copper) compared to D-DOPA or 1x PBS incubated cells.

Notes

  1. Prepare D-DOPA and L-DOPA solutions always fresh, just before the incubation of cells.
  2. Use the freshly formaldehyde fixed melanocytes for the assay.

Recipes

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4 (pH 7.4)
    Sterilized using 0.22 μm filtration system and stored at room temperature (25 °C)
  2. Acid wash and sterilization of coverslips
    1. Incubate the coverslips (approximately 100 in number) in 50 ml of HNO3 and HCl mixture (in 2:1 ratio) for 2 h at room temperature (25 °C).
    2. Wash the coverslips with deionized water for 3-4 times (50-100 ml each time) or until the pH of incubated water reached to 7.
    3. Wash the coverslips with 70% alcohol, dry in hot-air-oven (maintained at 50-60 °C) for overnight and sterilize by autoclaving in a glass beaker.
    4. Store the coverslips at room temperature in biosafety cabinet for repeated use.
  3. 0.1% Matrigel matrix solution
    1. Suspend 10 μl of Matrigel matrix in 10 ml of ice cold RPMI-1640 media (without fetal bovine serum) and keep the solution on ice during the experiment.
    2. Coat the coverslips by adding 1 ml of solution to each well at room temperature.
    3. After 1-2 min, collect the matrix solution and store at 4 °C for reuse.
    4. Dry the coverslips at room temperature for 15-30 min in a tissue culture cabinet with a closed lid.
  4. Growth media
    RPMI-1640 media
    10% fetal bovine serum
    1% L-Glutamine
    1% Penicillin-Streptomycin (antibiotic)
    Sterilized using 0.22 μm filtration system and stored at 4 °C
  5. 4% Formaldehyde solution
    Dilute 1.081 ml of Formaldehyde solution with 1x PBS to 10 ml and prepare the solution always fresh
  6. 0.1% DOPA solution
    Dissolve 10 mg of D- or L- DOPA in 10 ml of sterile 1x PBS and prepare the solutions always fresh

Acknowledgments

This protocol was modified from previously published reports (Zhao et al., 1994; Boissy et al., 1998). We thank E. V. Sviderskaya and D. C. Bennett for generous gift of wild type mouse melanocytes. This work was supported by Wellcome Trust-DBT India Alliance Senior Fellowship 500122/Z/09/Z to S. R. G. S. and a UGC fellowship 2120930821/2009 to R. A. J.

References

  1. Jani, R. A., Purushothaman, L. K., Rani, S., Bergam, P. and Setty, S. R. (2015). STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis. J Cell Sci 128(17): 3263-3276.
  2. Setty, S. R., Tenza, D., Sviderskaya, E. V., Bennett, D. C., Raposo, G. and Marks, M. S. (2008). Cell-specific ATP7A transport sustains copper-dependent tyrosinase activity in melanosomes. Nature 454(7208): 1142-1146.
  3. Sviderskaya, E. V., Kallenberg, D. M. and Bennett, D. C. (2010). The wellcome trust functional genomics cell bank: Holdings. Pigment Cell Melanoma Res 23: 147-150.

简介

黑素细胞在黑素体中产生黑色素,这些细胞器保护皮肤免受有害的紫外线。 酪氨酸酶是使用其底物氨基酸酪氨酸或L-DOPA(L-3,4-二羟基苯丙氨酸)引发颜料合成的关键铜酶蛋白酶。 此外,酪氨酸酶的活性与细胞色素沉着直接相关。 酪氨酸酶转运到黑素体中的缺陷或酶中的突变或降低的细胞内铜水平导致黑素体中酪氨酸酶活性的丧失,通常在白化病中观察到。 在这里,我们描述了一种方法来检测小鼠黑素细胞中酪氨酸酶的细胞内活性。 该协议将使存在于细胞内囊泡或细胞器(包括黑素体)中的活性酪氨酸酶可视化。

关键字:酪氨酸酶, 黑素小体, 眼皮肤白化病, 铜, 左旋多巴

材料和试剂

  1. 玻璃盖片(直径-12mm,No.1)(Polar Industrial Corporation,Blue Star,目录号:12mm圆形)
    注意:请参阅食谱的酸洗和消毒。
  2. 微载玻片(L-75mm×W-25mm×h-1.35mm)(Polar Industrial Corporation,Blue Star,目录号:PIC-1)
  3. 塑料组织培养(6孔)板(Corning,目录号:3506)和瓶顶真空过滤器(孔径0.22μm)(Corning,目录号:430015)
  4. 黑素细胞(永生野生型小鼠黑素细胞,来自C57BL/6J小鼠的黑色 - Ink4a-Arf-1,在此称为黑色 - Ink4a)[资源:The Wellcome Trust Functional Genomics Cell Bank(Sviderskaya et al。 >,2010)]
  5. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Sigma-Aldrich,目录号:C7631)
  6. 3,4-二羟基-D-苯丙氨酸(D-DOPA)(Sigma-Aldrich,目录号:D9378)
  7. 3,4-二羟基-L-苯丙氨酸(L-DOPA)(Sigma-Aldrich,目录号:D9628)
  8. HCl(Sigma-Aldrich,目录号:H1758)
  9. HNO 3(Merck Millipore,目录号:101799)
  10. KCl(Sigma-Aldrich,目录号:P9541)

  11. (Merck Millipore,目录号:104873)
  12. NaCl(Fisher Scientific,目录号:BP358-1)
  13. Na 2 HPO 4·2H 2 O(Fisher Scientific,目录号:S472-500)
  14. 乙醇(70%)(Merck Millipore,目录号:818760)
  15. 胎牛血清(Biowest,目录号:S1810-500)
  16. 甲醛(36.5-38%,在H 2 O中)溶液(HCHO溶液)(Sigma-Aldrich,目录号:F8775)
  17. Fluromount-G或封固剂(SouthernBiotech,目录号:0100-01)
  18. Matrigel Matrix(Corning Matrigel Growth Factor Reduced Basement Membrane Matrix,Phenol Red-Free)(Corning,目录号:356231)
  19. 青霉素 - 链霉素(抗生素)(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
  20. RPMI-1640培养基(Thermo Fisher Scientific,Gibco TM ,目录号:31800-022)
  21. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
  22. 0.1%DOPA溶液(参见配方)
  23. 4%甲醛溶液(见配方)
  24. 生长培养基(参见食谱)
  25. 0.1%Matrigel基质溶液(参见配方)
  26. 1x PBS(请参阅配方)

设备

  1. CO 2培养箱(保持在37℃,10%CO 2)(Thermo Fisher Scientific,型号:Forma Water Jacketed CO 2培养箱)
  2. 钳(通过高压灭菌器消毒)
  3. 明场显微镜(Olympus Corporation,型号:IX81电动倒置荧光显微镜)
  4. 热风炉(Eyela,目录号:NDO-420W)
  5. 玻璃烧杯(250ml)(Borosil,目录号:1000D21)

程序

  1. 准备盖玻片用于以下治疗:1×PBS和/或D-DOPA(阴性对照),L-DOPA和L-DOPA与铜。为此,使用镊子添加四个无菌盖玻片到6孔板的四个孔。
    注意:铜对于黑素细胞中的酪氨酸酶活性是必需的,并且向固定细胞中加入铜将允许在分泌区室例如内体和高尔基体中可视化酪氨酸酶活性,并增强其在黑素体中的活性(Setty等人,2008) 。
  2. 使用0.1%Matrigel基质溶液涂覆薄层的Matrigel盖玻片,并在室温下干燥盖玻片15-30分钟。
  3. 用1x PBS(每个2-3ml,室温)洗涤盖玻片三次。
  4. 在6孔板的每个孔中接种约0.5-0.8×10 6个小鼠黑素细胞或在任何培养皿中接种60-70%汇合,并在37℃下在10%CO 2中孵育,
  5. 24小时后用1%PBS(室温)中的4%甲醛固定细胞30分钟
  6. 用1×PBS(室温)洗涤细胞三次。
  7. 将细胞与以下新鲜制备的溶液在37℃孵育2小时 一个。 1×PBS和/或0.1%D-DOPA的1×PBS(阴性对照)
    b。 0.1%L-DOPA的1x PBS溶液中 C。在含有20μM硫酸铜(酪氨酸酶的辅助因子)的1×PBS中的0.1%L-DOPA
  8. 取出溶液,并与相应的溶液重复孵育。
    注意:L-DOPA在PBS或水中经历自动氧化,几个小时后变黑。此外,这种氧化导致溶液中活性L-DOPA底物的损失,因此,用新制备的溶液重复该步骤将改善酪氨酸酶的活性。
  9. 用1x PBS洗涤细胞三次。
  10. 使用安装介质将盖玻片安装在载玻片上,并在明场显微镜下成像(图1)
  11. 保持所有样品的明场曝光时间和设置相同(图1)。
  12. 比较L-DOPA或L-DOPA +铜处理的黑素细胞的明场图像与1×PBS或D-DOPA。可以分析图像的信号强度和颜料颗粒数量以及它们在细胞内的分布的差异。
    注意:用L-DOPA或L-DOPA +铜处理的黑色素细胞中颜料颗粒数量和强度的增加(与L-DOPA相比观察到更高的颗粒强度)表明在细胞内囊泡中存在活性酪氨酸酶,内吞细胞器包括黑素体(Setty等,2008)。

代表数据


图1. 体外 在野生型小鼠黑素细胞中的细胞内酪氨酸酶活性。在玻璃盖玻片上新鲜固定的小鼠黑素细胞用含有或不含硫酸铜(20μM)的1×PBS,D-DOPA(阴性对照)或L-DOPA孵育4小时。在倒置荧光显微镜下以明场模式在相同的相机设置下捕获图像。箭头表示用L-DOPA(有或没有铜)处理的细胞中增加的酪氨酸酶活性。箭头指向用L-DOPA +铜处理的细胞中具有增强的酪氨酸酶活性的区域。比例尺,10μm和插图,白色框区域的2.5倍。注意,与D-DOPA或1xPBS孵育的细胞相比,用L-DOPA(有或没有铜)处理的细胞中黑色素沉积物增加。

笔记

  1. 准备D-DOPA和L-DOPA溶液总是新鲜,只是在孵化细胞之前
  2. 使用新鲜甲醛固定的黑素细胞进行测定。

食谱

  1. 1x PBS
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH 2 PO 4(pH 7.4) 使用0.22μm过滤系统灭菌并在室温(25℃)下贮存
  2. 酸洗和盖玻片灭菌
    1. 在室温(25℃)下将盖玻片(约100个)在50ml HNO 3和HCl混合物(2:1比例)中孵育2小时。
    2. 用去离子水洗涤盖玻片3-4次(每次50-100ml)或直到孵育水的pH达到7。
    3. 用70%酒精洗涤盖玻片,在热风烘箱中干燥(保持在 ?50-60℃)过夜,并通过在玻璃中高压灭菌 烧杯。
    4. 将盖玻片在室温下储存在生物安全柜中重复使用。
  3. 0.1%Matrigel基质溶液
    1. 将10μlMatrigel基质悬浮于10 ml冰冷的RPMI-1640培养基中 (无胎牛血清),并将溶液保持在冰上 实验。
    2. 通过在室温下向每个孔中加入1ml溶液来涂布盖玻片。
    3. 1-2分钟后,收集基质溶液并储存在4°C下再次使用。
    4. 在封闭的盖子的组织培养箱中,在室温下将盖玻片干燥15-30分钟
  4. 生长介质
    RPMI-1640介质
    10%胎牛血清 1%L-谷氨酰胺 1%青霉素 - 链霉素(抗生素)
    使用0.22μm过滤系统灭菌并在4℃下贮存
  5. 4%甲醛溶液
    用1×PBS将1.081ml甲醛溶液稀释至10ml,并使溶液始终保持新鲜
  6. 0.1%DOPA溶液
    将10毫克D-或L-DOPA溶解在10毫升无菌1×PBS中,并准备溶液总是新鲜的

致谢

该协议从以前发表的报告(Zhao等人,1994; Boissy等人,1998)修改。我们感谢E.V.Sviderskaya和D.C.Bennett的野生型小鼠黑素细胞的慷慨礼物。这项工作得到Wellcome Trust-DBT印度联盟资深奖学金500122/Z/09/Z to S. R. G. S.和UGC fellowship 2120930821/2009 to R. A. J.

参考文献

  1. Jani,R.A.,Purushothaman,L.K.,Rani,S.,Bergam,P.and Setty,S.R。(2015)。 STX13在黑素体生物发生过程中调节来自回收内体的货物运输。细胞科学 128(17):3263-3276。
  2. Setty,S.R.,Tenza,D.,Sviderskaya,E.V.,Bennett,D.C.,Raposo,G.and Marks,M.S.(2008)。 细胞特异性ATP7A转运维持黑素体中铜依赖性酪氨酸酶活性。 自然 454(7208):1142-1146。
  3. Sviderskaya,E.V.,Kallenberg,D.M.and Bennett,D.C。(2010)。 Wellcome trust功能基因组细胞库:控股。 Pigment Cell Melanoma Res 23:147-150。
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引用:Jani, R. A., Nag, S. and Setty, S. R. (2016). Visualization of Intracellular Tyrosinase Activity in vitro. Bio-protocol 6(8): e1794. DOI: 10.21769/BioProtoc.1794.
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